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1 Institute of Molecular Biology, University of Zurich, 8057 Zurich, Switzerland; 2 Ph.D. Program in Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland; 3 Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA; 4 Invitrogen Corporation, Carlsbad, California 92008, USA
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[Keywords: Apoptosis; GLA-3; MAPK signaling; germline development; C. elegans ]
Received February 24, 2006; revised version accepted June 1, 2006.
; Bender et al. 2002). Germ cell development is also characterized by either massive waves or low but constant levels of apoptosis that are not caused by genetic defects. For example, >99.9% of oocytes undergo apoptosis in response to hormonal changes that occur at several stages during the female life cycle in mammals (Tilly 2001; Kim and Tilly 2004). Apoptosis is also the fate of 
50% of germ cells undergoing oogenesis in the gonad of Caenorhabditis elegans hermaphrodites. (Gumienny et al. 1999). Characterization of the pathways that regulate germ cell death will contribute to our understanding of the cell suicide decision and might allow for more efficient therapeutic manipulation of the apoptotic program.
C. elegans is a good model to study the signaling cascades involved in the decision between germ cell survival and germ cell death (Hengartner 1997; Gumienny et al. 1999). The adult hermaphrodite gonads consist of two U-shaped tubes that are connected at a common uterus. At the distal end of each gonad, mitotic germ stem cells proliferate in response to the Notch ligand LAG-2. Cells beyond the influence of LAG-2 enter meiosis and progress through the pachytene stage of meiosis I; this transition requires activation of the RAS/MAPK (MAP kinase) signaling cascade (Hubbard and Greenstein 2000; Seydoux and Schedl 2001). Following transition through pachytene, germ cells can either enter diakinesis of meiosis I and differentiate into oocytes or undergo apoptosis. We previously suggested that these cell deaths are the result of a physiological, homeostatic control mechanism that limits the number of germ cells permitted to differentiate into oocytes (Gumienny et al. 1999).
During C. elegans somatic development, apoptosis is triggered when the pro-apoptotic BH3-domain-containing protein EGL-1 interacts with the BCL-2 family member CED-9 on the surface of mitochondria. Binding of EGL-1 induces CED-9 to release the sequestered Apaf-1 homolog CED-4, resulting in the formation of a C. elegans apoptosome and activation of the caspase CED-3 (Chen et al. 2000; Yan et al. 2004, 2005; for review, see Lettre and Hengartner 2006). We previously showed that germline apoptosis can be induced by both p53-dependent and p53-independent pathways (Lettre et al. 2004). However, whereas DNA damage-induced germline apoptosis depends on the activation of EGL-1, physiological germline apoptosis occurs normally in the absence of EGL-1, indicating that additional regulatory factors are required to trigger the apoptotic cascade in these cells (Gumienny et al. 1999; Gartner et al. 2000).
In an attempt to identify genes that regulate physiological germ cell apoptosis, we performed a forward genetic screen to isolate mutants with increased levels of germline apoptosis. In this paper, we report the cloning and characterization of gla-3 (germline apoptosis), a gene that encodes a predicted RNA-binding protein of the TIS11 family. Loss of gla-3 function results in increased germ cell apoptosis and severe defects in oocyte differentiation, which lead to reduced brood size. Biochemical analysis revealed that GLA-3 physically interacts with the C. elegans MAPK MPK-1, providing a direct link between the apoptotic process and oogenesis. Furthermore, we show that GLA-3 functions as an inhibitor of the MAPK cascade during vulva formation and in muscle cells. Our findings indicate that GLA-3 is a new component of the MAPK signaling cascade and highlight a molecular link between germ cell survival and pachytene progression in the C. elegans germline.
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Results |
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To identify novel genes involved in the regulation of germ-cell apoptosis, we performed a forward genetic screen to isolate mutations that result in increased germline apoptosis, as described in Materials and Methods. gla-3 was selected for cloning and further characterization based on the severity of its germline apoptosis phenotype. All three gla-3 alleles that we analyzed (op212 and op216, isolated in our screen, and ep312, generously provided by M. Costa, Exelixis) resulted in increased numbers of germ cell corpses that displayed typical apoptotic morphology (Fig. 1A,B). Because gla-3 mutants exhibited normal patterns of somatic cell death (data not shown), we conclude that gla-3 is not a general cell-death regulator.
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). Whereas inactivation of the engulfment gene ced-1 largely abolished YFP::actin staining, most apoptotic germ cells in gla-3(lf); opIs110 (lf, loss of function) animals were surrounded by a YFP::actin halo, indicating that the cells were being engulfed efficiently (Supplemental Fig. 1). These results support our notion that loss of gla-3 function results in increased germ cell death, rather than decreased cell corpse clearance. To determine an epistatic relationship between gla-3 and other components of the apoptotic machinery, we generated strains that contain gla-3(op212) and either of the strong loss-of-function mutations ced-4(n1162) or ced-3(n717). In both double mutants, germ cell death was completely abrogated, demonstrating that gla-3(lf)- induced cell death is apoptotic in nature and that the components of the apoptotic machinery function downstream of gla-3 (Fig. 1C).
Because germline apoptosis can be mediated by both DNA damage-dependent and -independent mechanisms (Gartner et al. 2000), we carried out epistasis analysis between gla-3(op212) and genes that are known to be involved in DNA damage responses. hus-1(op244) and clk-2(mn159ts) mutants are characterized by a complete absence of DNA damage-induced apoptosis, but have nearly normal levels of somatic and germline apoptosis (Ahmed et al. 2001; Hofmann et al. 2002). gla-3(op212); clk-2(mn159ts) and hus-1(op244) gla-3(op212) worms had similar levels of germline apoptosis as gla-3(op212) alone, indicating that gla-3-induced apoptosis occurs independently of these DNA damage checkpoint genes (Fig. 1D). Because DNA damage-induced apoptosis is dependent on the p53 homolog cep-1 (Derry et al. 2001; Schumacher et al. 2001), we inactivated gla-3 using RNA-mediated interference (RNAi) in cep-1(gk138) mutants. Consistent with our previous findings (Lettre et al. 2004), we observed similar levels of germline apoptosis in gla-3(RNAi) cep-1(gk138) and in gla-3(RNAi) alone (Fig. 1E), showing that loss of gla-3 does not activate a DNA damage apoptotic response.
Finally, we tested the ability of gla-3 mutants to respond to exogenous DNA damage. We previously showed that ionizing radiation induces apoptosis of meiotic cells and transient cell cycle arrest of the mitotic germ cell population (Gartner et al. 2000). Both apoptosis and cell cycle responses appeared normal in gla-3(op212) mutants (Fig. 1D; Supplemental Fig. 2, respectively). These data suggest that the DNA damage response pathways are functional in gla-3 mutants, and are consistent with a role of gla-3 in the physiological cell-death pathway.
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We genetically mapped gla-3(op212) to a 0.36-cM interval, on the right arm of Chromosome I, between the single nucleotide polymorphism markers pk1058 at 2.31 cM and opP106 at 2.67 cM, as described in Materials and Methods. We inactivated the 40 genes predicted to reside between these two markers using RNAi, in the hope that knockdown of the relevant gene would phenocopy gla-3(op212). A single gene, T02E1.3, which encodes two splice variants, gave rise to increased germline apoptosis when inactivated by RNAi. We sequenced T02E1.3 from wild-type, op212, and op216 animals and found a C-to-T transition at position 471 in T02E1.3a (position 574 in T02E1.3b) in op212, resulting in a premature stop codon (Fig. 2A), whereas op216 mutants contain a G-to-A transition in the acceptor splice site of the first intron of T02E1.3a (Fig. 2A). The third allele of gla-3, ep312, was generated in an independent screen for mutants with increased germline apoptosis in a cep-1(lf) mutant background (M. Costa, pers. comm.). ep312 results in a 265-bp deletion that removes part of the last two exons of gla-3 and introduces a frameshift at the new junction (Fig. 2A). Based on the strength of its phenotype and the molecular nature of the mutation, we believe op212 to be a null allele of gla-3.
gla-3 encodes a TIS11-like zinc finger domain protein
gla-3 encodes a protein that contains two CCCH-like zinc-finger domains (Fig. 2B). This domain is found in a subset of zinc-finger family proteins; its consensus sequence corresponds to C-X8–10-C-X5-C-X3-H, where X refers to any amino acid (Varnum et al. 1989). CCCH zinc fingers were first described in the mouse TIS11 protein, where they are responsible for nucleic-acid binding (Varnum et al. 1989; DuBois et al. 1990; Bai and Tolias 1996). Mammalian TIS11-like zinc-finger-containing proteins have been shown to function either as transcription factors or RNA-binding proteins (Taylor et al. 1996; Worthington et al. 2002). Several C. elegans genes encoding proteins with TIS11-like zinc-finger domains have been described, including pie-1, mex-1, pos-1, mex-5, and mex-6, all of which are involved in early blastomere cell-fate determination and germline development (Mello et al. 1996; Guedes and Priess 1997; Tabara et al. 1999; Schubert et al. 2000). In addition, the recently identified TIS11-like proteins OMA-1, OMA-2, and OMA-3 are redundantly required for prophase progression during oocyte maturation (Detwiler et al. 2001; Shimada et al. 2002). GLA-3 is highly similar to these proteins in the CCCH zinc-finger domain (Fig. 2B), but bears no significant sequence similarity to other characterized proteins outside of the zinc fingers. Unlike most of the characterized CCCH-zinc-finger-containing proteins in C. elegans, the zinc-finger domains in GLA-3 are situated at the two ends of the protein, 400 amino acids apart.
gla-3 is expressed in the germline
We probed a developmental Northern blot from wild-type animals with a probe hybridizing to both gla-3 transcripts. We found gla-3 to be expressed in embryos, L4-stage larvae, and adults (Fig. 3A), with the highest expression being in L4 larvae. The predominant developmental difference between an L4 larva and an adult hermaphrodite is the expansion of the germline. To examine the expression of gla-3 in the adult hermaphrodite germline, we prepared RNA from three temperature-sensitive mutants, defective in various aspects of germline development. At the nonpermissive temperature, glp-4(bn2ts) mutants produce a somatic gonad that is largely devoid of germ cells (Beanan and Strome 1992), fem-2(e2105ts) mutants make oocytes but no sperm, and fem-3(q20sd) mutants make sperm but no oocytes (Barton et al. 1987). We detected gla-3 expression in all three mutant backgrounds, demonstrating that gla-3 is expressed in both germline and soma (Fig. 3B). Very low levels of mRNA were observed in gla-3(op212) mutants, consistent with the finding that this mutation causes a premature stop, which often results in rapid elimination of the mutant transcript via nonsense-mediated decay (Weischenfeldt et al. 2005).
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). GFP expression was observed throughout the germline from the L4 stage onward and in all cells of the early embryo (Fig. 3C), consistent with the gla-3 mRNA pattern in the developmental Northern blot (Fig. 3A). Interestingly, expression was also observed in muscle cells in threefold stage embryos (Fig. 3C, panel c). To further investigate temporal and spatial gla-3 expression, we raised specific polyclonal antibodies against peptides derived from the unique N termini of GLA-3A (T02E1.3a) and GLA-3B (T02E1.3b). Western blot analysis of synchronized wild-type worms revealed that both isoforms are expressed at the L4 and adult stages, whereas no expression was detectable from the L1 to L3 stages, nor in gla-3(op212) mutants (Fig. 3D). To examine the expression pattern of GLA-3 in the germline, we investigated the localization of the GLA-3 protein by in situ immunostaining. Consistent with the mRNA expression pattern, GLA-3B was detectable in L4 and adult gonads. The protein was expressed in the mitotic and meiotic zones of the adult germline, and localized to the cytoplasmic compartment, with an enrichment in cortical/submembrane regions (Fig. 3E); a similar pattern of expression was observed with GLA-3A (data not shown). The GLA-3B staining was absent in the gonads of gla-3(op212) mutants (Fig. 3E).
Loss of gla-3 results in impaired oocyte development
gla-3(lf) mutants were morphologically normal, but exhibited a greatly reduced brood size and a low frequency of embryonic lethality (Table 1). Both defects were milder in gla-3(op216) mutants, consistent with the predicted hypomorphic nature of this mutation (which affects only one of the two GLA-3 isoforms). All three mutants showed a further reduction in brood size when raised at high temperature (25°C) (Table 1). Because this reduction was also apparent in the predicted null mutant gla-3(op212), we surmise that loss of gla-3 function uncovers an intrinsically temperature-sensitive process that is required for oocyte development.
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Consistent with the above conclusion, the gonads of gla-3(op212) adult hermaphrodites contained few, abnormally shaped oocytes (Fig. 4A). To examine whether the reduced brood size was the result of excessive apoptosis, we observed gonads of gla-3(op212); ced-3(n717) double mutants. Although apoptosis was completely suppressed in these animals (Figs. 1C, 4A), oogenesis was still impaired, indicating that gla-3 function is necessary for oocyte differentiation rather than germ cell survival per se (Table 1).
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Inhibition of apoptosis did not significantly improve the differentiation defects that are caused by loss of gla-3 function: DAPI staining of gla-3(op212); ced-3(n717) double mutants revealed an unusually large number of pachytene-stage nuclei in the proximal arm (Fig. 4; data not shown). The double mutant did contain more diakinesis stage oocytes than the gla-3 single mutant; however, these diakinetic oocytes were misshapen and abnormally small. These results indicate that inhibition of apoptosis allows more female germ cells to enter diakinesis in gla-3 mutants, but does not rescue the gla-3(lf) differentiation defect(s).
Biochemical interaction of GLA-3 and MPK-1 in vitro and in vivo
To identify molecular links between GLA-3 and other proteins involved in oogenesis, we searched the worm interactome (WI5, http://vidal.dfci.harvard.edu/interactomedb) for interaction partners of GLA-3, and found a yeast two-hybrid (Y2H) interaction between GLA-3 and the two isoforms of MPK-1. Using these two MPK-1 iso-forms as baits, both GLA-3 isoforms were found as the predominant preys (over 50% of hits) in the AD-cDNA and AD-ORFeome prey libraries (Li et al. 2004; data not shown). MPK-1 is the C. elegans homolog of the mammalian ERK1/2 serine/threonine kinases. MPK-1 is required for germline development: In mpk-1(lf) mutants, germ cells fail to exit the pachytene stage of meiosis I, resulting in sterility and decreased germ cell apoptosis (Church et al. 1995; Lackner and Kim 1998; Gumienny et al. 1999).
To verify the Y2H result, we performed glutathione-S-transferase (GST) affinity purification experiments in 293T cells transfected with MPK-1 fused to GST and myc-tagged GLA-3. Both isoforms of GLA-3 interacted with both isoforms of MPK-1 (Fig. 5A). To validate the in vitro evidence and show that MPK-1 interacts with GLA-3 in vivo, we immunoprecipitated GLA-3 from C. elegans extracts and tested for MPK-1 binding. As illustrated in Figure 5B, MPK-1 could be immunoprecipitated with both isoforms of GLA-3. Our findings show a direct interaction between GLA-3 and MPK-1 in vitro and in vivo, and provide a possible explanation for the germline defects observed in gla-3(lf) mutants.
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). In addition, GLA-3 contains potential MPK-1 binding sites, as defined by a cluster of positive amino acids surrounded by hydrophobic amino acids (Tanoue and Nishida 2002). GLA-3a has one site at position 66–74 (LRKVVRIDR), whereas GLA-3b has two such sites, at positions 66–74 (LRKTKI) and 99–107 (LRKVVRIDE). Taken together, our biochemical and molecular data are consistent with a role of GLA-3 as a regulator or a target of MPK-1 in the C. elegans germline.
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Do ras/MAPK pathway genes regulate the apoptotic program? Strong loss-of-function mutations in the mpk-1 pathway cause a sterile phenotype due to developmental arrest at the pachytene stage of meiosis (Church et al. 1995). We and others have previously shown that pachytene-arrested cells do not undergo apoptosis (Gumienny et al. 1999; Navarro et al. 2001). To address the role of mpk-1 in gla-3(lf)-induced cell death, we performed genetic epistasis analysis between gla-3 and genes that are known to be involved in the Ras/MAPK pathway. Using the putative null mutation mpk-1(ga117) (Lackner and Kim 1998), we found that gla-3(op212)-induced apoptosis was completely abrogated in gla-3(op212); mpk-1(ga117) double mutants (Fig. 7A). Interestingly, although apoptosis is suppressed, gla-3 does not rescue the sterile phenotype of mpk-1(ga117) (Supplemental Table II). These data demonstrate that mpk-1 is epistatic to gla-3, and that its activation is required for gla-3(lf)-induced germ cell death. We then compared the levels of germ cell death in mpk-1(oz140) ced-9(n2812) adult hermaphrodites with those in gla-3(RNAi); mpk-1(oz140) ced-9(n2812) animals. ced-9(lf)-induced and gla-3(RNAi)-induced germ cell apoptosis were both completely suppressed in the absence of functional mpk-1 (Gumienny et al. 1999; Supplemental Table III).
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), resulted in increased germline apoptosis, with levels comparable to gla-3 mutants (Fig. 7B). Similarly to gla-3 mutants, lip-1(zh15)-induced germline apoptosis depended on ced-3 but was cep-1-independent (Supplemental Fig. 3), indicating that these two genes might function in the same genetic pathway. Indeed, gla-3(op212); lip-1(zh15) double mutants showed only a slight increase in germline apoptosis over the single mutants (Fig. 7B).
lip-1(zh15) mutants display accelerated oocyte development due to defective G2/M-phase arrest (Hajnal and Berset 2002). We scored the number of diakinetic oocytes in the gonads of single and gla-3(op212); lip-1(zh15) double mutants and found that the double mutants exhibited decreased numbers of diakinetic oocytes, a phenotype similar to gla-3(op212) single mutants (Supplemental Table II). These observations suggest that gla-3 is epistatic to lip-1 for germ cell differentiation.
gla-3 mutations do not result in MPK-1 overactivation
To test whether loss of gla-3 function altered the pattern of MPK-1 activation in the germline, we stained dissected gonads with a monoclonal antibody that specifically recognizes the diphosphorylated, activated form of MPK-1 (DP-MPK-1). As previously reported (Miller et al. 2001; Page et al. 2001), MPK-1 was activated in pachytene-stage cells and also, in response to the sperm signal, in the first and sometimes second oocyte proximal to the spermatheca (Supplemental Fig. 4A). The pattern of MPK-1 activation in gla-3(op212) mutants was similar to that of wild-type animals, although the DP-MPK-1 staining in the oocytes proximal to the spermatheca was occasionally weaker (Supplemental Fig. 4A), possibly reflecting the abnormal nature of oocytes in gla-3 mutants.
To further test the hypothesis that GLA-3 might regulate the levels of activated MPK-1, we used an in vitro MPK-1 kinase assay (Alessi et al. 1995; Berset et al. 2001). Using myelin basic protein (MBP) as an MAPK substrate, we found that both lip-1(zh15) and let-60(n1046gf) animals had higher levels of MAPK enzymatic activity when compared with the wild type (Supplemental Fig. 4B). In contrast, the level of MAPK activity in gla-3(op212) and gla-3(op216) mutants was similar to the wild type (Supplemental Fig. 4B). Taken together, these data indicate that inactivation of GLA-3 does not directly affect the activation of MPK-1 protein itself.
GLA-3 is a negative regulator of the MAPK signaling pathway in somatic tissues
During C. elegans development, LET-60/Ras and MPK-1/MAPK transmit the LIN-3/EGF inductive signal in vulval precursor cells (VPCs), resulting in vulva formation (Moghal and Sternberg 2003). Loss-of-function mutations in genes that mediate the inductive signaling pathway can lead to a vulvaless (Vul) phenotype due to insufficient induction of VPCs. Conversely, mutations that enhance the inductive signal result in a multivulva (Muv) phenotype, due to hyperinduction of VPCs.
To test the possibility that GLA-3 might also influence MAPK signaling during vulval development, we examined double mutants between gla-3(lf) and various mutations that affect vulval induction. In gla-3(lf) single mutants, vulval development appeared normal (Table 3). However, loss of gla-3 function enhanced the multivulva phenotype observed in two different let-60(gf) mutants (Table 3), suggesting that GLA-3 might function as an inhibitor of MAPK signaling in VPCs. Similarly, vulval development is normal in lip-1(lf) mutants (Berset et al. 2001), whereas double gla-3(op212); lip-1(zh15) mutants displayed a weak synthetic Muv phenotype (Table 3). In contrast, the gla-3 alleles could not suppress the mpk-1(ga117) vulvaless phenotype. Taken together, these results indicate that GLA-3 might function as an inhibitor of MAPK signaling during vulval development.
Activation of the Ras/MAPK pathway has also been shown to promote protein degradation in C. elegans muscles, leading to a progressive loss of motility (Szewczyk et al. 2002). We therefore investigated whether gla-3 might influence muscle-protein degradation through its interaction with the Ras/MAPK pathway. Whereas gla-3 mutants raised at 15°C showed nearly wild-type movement, animals shifted to 25°C at the L4 stage suffered a progressive loss of motility, similar to the defect observed in let-60(ga89) mutants (Table 4). The locomotion defect was not significantly enhanced in the gla-3(lf); let-60(lf) double mutants, implying that the two genes might affect the same molecular process.
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; Szewczyk et al. 2000). This fusion protein is completely stable in well-fed wild-type worms; therefore, any subsequent decline in LacZ activity is the result of protein degradation. Well-fed gla-3(op212) mutants exhibited wild-type levels of LacZ activity when grown at 15°C. In contrast, mutants shifted to 25°C at the L4 stage showed a time-dependent loss of reporter activity similar to the loss observed in mutants with an overactivated Ras/MAPK pathway (Fig. 8). Taken together, these observations indicate that GLA-3 might negatively regulate Ras/MAPK-dependent protein degradation in muscle cells.
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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), the factors that regulate germline apoptosis are largely unknown. In an attempt to isolate such regulators, we performed a genetic screen and identified gla-3. We found that gla-3 is important for oocyte differentiation and germ cell survival and encodes a TIS11-like zinc-finger-containing protein. We showed that GLA-3 genetically and physically interacts with the C. elegans MAPK MPK-1 and provided evidence to support the notion that GLA-3 might function as a negative regulator of the MAPK pathway in the developing vulva and in muscle cells.
Regulation of germline apoptosis by GLA-3 and other RNA-binding proteins
We showed that gla-3 has two isoforms, which differ in their two 5'-most exons. Are both isoforms involved in germline apoptosis and oocyte development? Both GLA-3A and GLA-3B are expressed in the germline, and both isoforms interact with MPK-1. Furthermore, the op216 mutation, which is predicted to affect only GLA-3A, shows weaker defects than the predicted null alleles. Based on these results, we suggest that both isoforms might participate in germline development and apoptosis. However, further work is required to confirm this hypothesis, and to determine whether the isoform-specific N-terminal domains perform distinct functions.
How could GLA-3 regulate germ cell apoptosis? The TIS11-like proteins that have previously been characterized in C. elegans, including PIE-1, POS-1 MEX-1, MEX-5, and MEX-6, function in a hierarchical regulatory cascade that controls cell fate during early embryonic development (Schneider and Bowerman 2003). In addition, the combined loss of the TIS11-like proteins, OMA-1 and OMA-2, results in impaired oocyte maturation and female infertility (Detwiler et al. 2001). The mammalian TIS11 zinc-finger-containing protein, tristetraprolin (TTP), is an RNA-binding protein that interacts with 3'-untranslated AU-rich elements of mRNAs and negatively regulates their expression (Lai et al. 1999; Carballo et al. 2000; Cao et al. 2003). Based on the homology between the C. elegans and the mammalian TIS11-like proteins, it is possible that PIE-1, POS-1, MEX-1, MEX-5, MEX-6, and GLA-3 exert their functions by acting as mRNA-binding proteins (Barabino et al. 1997; Lai et al. 2000; Worthington et al. 2002).
Inactivation of several RNA-binding proteins, including DAZ-1, CGH-1, CPB-3, and CAR-1, has previously been shown to trigger apoptosis in the C. elegans germline (Karashima et al. 2000; Navarro et al. 2001; Lettre et al. 2004; Boag et al. 2005). Similarly to gla-3, daz-1 mutations result in severe oogenic defects due to arrest at the pachytene stage of meiosis I. It is possible that these genes coordinately regulate the synthesis of pro-apoptotic or survival proteins during germline development. Indeed, CGH-1 and CAR-1 interact physically and are components of an evolutionarily conserved RNP complex (Boag et al. 2005). Alternatively, these proteins might function more broadly to promote germline differentiation, and the developmental defects that result from their loss could be the cause of the apoptotic phenotypes observed. Identification of the target mRNAs regulated by these various proteins is required to shed light on this issue.
GLA-3 is a novel component of the MAPK signaling cascade
In addition to its potential function as an RNA-binding protein, GLA-3 also interacts physically with the C. elegans MAPK MPK-1 in vitro and in vivo, indicating that GLA-3 might have a role in MPK-1 signaling. In the hermaphrodite germline, activation of the LET-60/MEK-2/ MPK-1 signaling pathway is essential for pachytene progression and subsequent entry into diplotene/diakinesis (Church et al. 1995; Lackner and Kim 1998; Miller et al. 2001; Page et al. 2001). Although MPK-1 activation during pachytene progression appears to be transient and tightly regulated (Hajnal and Berset 2002), the nature of the signal that controls its activation and the downstream effectors of MPK-1 remain poorly characterized. Based on its mutant phenotype and its physical interaction with MPK-1, GLA-3 might be directly involved in this process.
Because our expression studies revealed that GLA-3 expression is not restricted to the germline, we also studied the potential role of GLA-3 in MPK-1 signaling in the soma. We found that the absence of gla-3 resulted in phenotypes consistent with an inappropriate activation of mpk-1 signaling in both vulval development and protein degradation in body wall muscles. These biochemical and genetic analyses support the idea that GLA-3 functions as a negative component of the Ras/MAPK pathway. However, we were so far unable to detect any change in MPK-1 activity in gla-3 mutants or worm extracts (Supplemental Fig. 4), suggesting that GLA-3 does not directly regulate MPK-1 activation but, rather, might restrict its access to or interaction with substrates.
Could GLA-3 be a substrate rather than—or in addition to—a regulator of MPK-1? Neither our genetic nor our biochemical data allow us to definitively exclude this possibility. We did not observe any differences in the GLA-3 protein levels in different mutants of the MAPK pathway by Western blot analysis and in situ immunostaining (Supplemental Fig. 5; data not shown), which indicated that MPK-1 does not directly regulate GLA-3 levels. However, it is possible that MPK-1 phosphorylates GLA-3 and this modification is important for GLA-3 function. Indeed, several kinases, including MAPKMAP kinase 2 (MK2), ERK2, p38, and JNK, can phosphorylate the mammalian TIS11 protein tristetraprolin in response to growth factor or cytokine treatment (Taylor et al. 1995; Mahtani et al. 2001; Chrestensen et al. 2004). Although GLA-3 has several potential MAPK phosphorylation sites, we were unable to determine whether GLA-3 is phosphorylated by MPK-1 in vivo using the antibody that we raised (Supplemental Material 5).
What is the function of GLA-3 in the MAPK signaling pathway?
In Schizosaccharomyces pombe, MAPK signaling activity can be regulated through a negative feedback loop that involves Rnc1, a KH-domain-containing RNA-binding protein that stabilizes the Pmp1 phosphatase mRNA (Sugiura et al. 2003). Based on the ability of GLA-3 to bind to MPK-1 and its predicted RNA-binding domains, a similar model could be envisaged in which GLA-3 is activated through interaction with MPK-1, and in response stabilizes mRNAs encoding negative regulators of MPK-1 such as LIP-1.
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Materials and methods |
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Maintenance and genetic manipulation of C. elegans were carried out as described (Brenner 1974). The following mutations and transgenes were used: LG I: hus-1(op244), gla-3(op212), gla-3(op216), gla-3(ep312), cep-1(gk138), glp-4(bn2ts); LG II: lip-1(zh15); LG III: fem-2(e2105ts), mpk-1(ga117), mpk-1(oz140), ced-4(n1162), clk-2(mn159ts), unc-32(e189), ced-9(n2812), unc-119(ed3), opIs64[Pgla-3a::2xNLS::gfp unc-119(+)]; LG IV: fem-3(q20sd), ced-3(n717), let-60(n1046gf), let-60(ga89gf), opIs110[Plim-7::yfp::act-5 unc-119(+)]; LG V: ccIs55[unc-54::lacZ; sup-7(st5)], gaIs36[HS-mpk-1(+) EF1-D-mek(+) unc-30(+)]. All mutations are described in WormBase (http://www.wormbase.org). The Bristol strain N2 was used as the wild-type strain. Strains were maintained at 20°C unless stated otherwise. Unless specified otherwise, all phenotypic characterizations were performed on hermaphrodites 24 h post-L4/adult molt. Movement rates were measured as described (Bolanowski et al. 1981).
Isolation of gla-3 alleles
gla-3(lf) alleles were isolated in an F 2 screen for mutants with increased germ cell apoptosis. Wild-type animals were mutagenized with ethylmethane sulfonate using a standard protocol (Brenner 1974), and adult F 2 animals with increased apoptotic germ cell corpses were identified with the vital dye acridine orange (AO; Sigma) (Lettre et al. 2004). Two independent alleles (op212 and op216) were isolated from a screen of 45,000 genomes. To eliminate possible additional mutations, each mutant was crossed back to the wild type eight times before subsequent analysis. The ep312 deletion allele was isolated in a separate screen, performed to identify mutants involved in cep-1-independent germ cell apoptosis (M. Costa, pers. comm.).
Cloning of gla-3
The op212 allele of gla-3 was mapped near the middle of Chromosome I by two-factor mapping using the marker dpy-5. To refine the position of gla-3, three-factor mapping was conducted using the following marker combinations: dpy-5 unc-87, dpy-5 unc-29, and dpy-24 unc-75. This analysis placed gla-3 closer to unc-29 than dpy-5. SNP mapping refined the region to 0.36 cM (roughly 270 kb), which included 40 predicted genes. We then performed RNAi feeding of the 40 genes in this interval. Only inactivation of T02E1.3 resulted in increased germline apoptosis. The molecular changes induced by the op212, op216, and ep312 mutations were determined by PCR amplification of the gla-3 locus from the respective mutants, followed by sequencing of both strands of the amplification product.
RNA interference
Feeding RNAi was performed as previously described (Lettre et al. 2004). Feeding RNAi constructs were obtained either from the Ahringer library (Kamath et al. 2003) or from the ORFeome RNAi collection (Rual et al. 2004), or were generated by sub-cloning from cDNAs (for bir-1 and gfp).
Germline apoptosis
Apoptotic germ cell corpses were identified and quantitated based on their characteristic morphology under differential interference contrast (DIC) optics, as previously described (Gumienny et al. 1999).
Brood size and oocyte counts
Brood size counts were performed as previously described (Hofmann et al. 2002). Oocyte counts were performed according to Hajnal and Berset (2002).
Northern analysis
Total RNA was isolated using TRIZOL reagent (GIBCO-BRL) from cultures of synchronized wild-type worms at different developmental stages as well as from gla-3(op212), glp-4(bn2ts), fem-2(e2105ts), and fem-3(q20sd) adults. Poly(A)+ selection of mRNAs from staged adults was performed using the Micro-PolyA kit (Ambion). RNA samples were subjected to formaldehyde agarose gel electrophoresis, transferred to Hybond N+ nylon membrane, and probed with a full-length gla-3 cDNA amplified from plasmid yk6h11 and labeled with 32P using the random hexamer primer method.
Generation of GLA-3 antibodies
Rabbit polyclonal antibody T02E1.3a (1301) was raised against the T02E1.3a peptide KTQEISVVIDPRDA, whereas antibody T02E1.3b (1297) was raised against the T02E1.3b peptide LLNSDMDPVRNLES. Peptides were synthesized (Sigma-Genosys) and injected into two rabbits according to Sigma protocols.
Western blot analysis
Adult worms were washed off the plates with water. Worm pellets were frozen in liquid nitrogen and kept at –80°C. To extract proteins, an equal volume of acid-washed glass beads (Sigma) was added to the frozen worm pellet, and the tubes were put in a bead beater (FastPrep FP120) for 35 sec at speed 6.5. Following addition of 100–300 µL of RIPA buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 1% SDS, 1 tablet/10 mL Complete Mini protease inhibitor [Roche]) or immunoprecipitation buffer (25 mM HEPES-NaOH at pH 7.4, 150 mM NaCl, 0.2 mM DTT, 10% glycerol, 1% Triton X-100, 1 tablet/10 mL Complete Mini protease inhibitor [(Roche]), tubes were vortexed and incubated on ice for 10 min. Extracts were centrifuged for 15 min at 13,000 rpm at 4°C. Supernatants were collected and kept at –80°C. Protein quantification was done using the Bio-Rad protein assay as recommended, with bovine serum albumin (BSA) as standard. The antibody dilutions used were 1:1000 for GLA-3 antibodies and 1:10,000 for 
-tubulin (Upstate). Secondary HRP-labeled anti-rabbit and anti-mouse antibodies (Sigma) were used at 1:100,000 and 1:40,000, respectively, and detected using ECL (Sigma).
Transgenic worms
The Pgla-3a::2xNLS::GFP unc-119(+) construct (pEK1) was bombarded into unc-119(ed3) worms as previously described (Praitis et al. 2001; Hofmann et al. 2002). Integration of each construct was determined by loss of visible Unc-119 offspring. At least three integrated lines were generated; all showed the same expression pattern.
Reverse two-hybrid selection
The system we used is a variation of the original reverse two-hybrid protocol developed by Vidal (Vidal et al. 1996a, b). Briefly, the mpk-1 wild-type ORF was mutagenized by PCR using 30 amplification cycles and Platinum Taq DNA polymerase (Invitrogen). To build the mpk-1 mutant library, the gel-purified PCR product was cloned into the pDONR-Express vector using the Gateway in vitro cloning system (Invitrogen). In this vector, the ORF is cloned under control of an IPTG-inducible promoter in frame with the KanR gene. Clones containing PCR-induced STOP mutations are removed by plating TOP-10 (Invitrogen) bacteria transformants on IPTG (1 mM) and Kan (80 µg/mL) selective medium. About 500,000 independent transformants were obtained. The ORFs were LR cloned into the pPC-97 Gal-4 DNA-binding domain yeast two-hybrid vector and retransformed into electrocompetent TOP-10 cells. This library was transformed into MaV203 yeast cells containing GLA-3 fused to the Gal-4 activation domain. Cotransformed cells were selected for their ability to grow on –Leu, –Trp, +5-FOA (5-fluoro-orotic acid) medium, and the mpk-1 ORF was amplified by PCR and sequenced so that the causative mutation could be identified. These mpk-1 mutants were retested by gap repair in fresh MaV203 containing the AD GLA-3 fusion, to confirm that the interaction had been disrupted because of the mutation (Walhout and Vidal 2001).
Visualization of amino acid changes on MPK-1
Mutations identified in mpk-1 using the yeast reverse two-hybrid system were mapped to the structure model of MPK-1 (SWISS-PROT P39745
[GenBank]
), which was derived from the rat ERK2 (SWISS-PROT P63086
[GenBank]
) crystal structure (Zhang et al. 1993, 1994). The location of the changed amino acids was visualized on the 3D structure using Swiss-PdbViewer (Guex and Peitsch 1997).
GST pull-downs
mpk-1a or mpk-1b ORFs were cloned into pDEST-27, which contains GST coding sequence upstream of the Gateway recombination site (Invitrogen). gla-3a and gla-3b ORFs were cloned into pDEST-CMV-myc, which contains the myc tag upstream of the Gateway recombination site (Invitrogen). Both vectors use the CMV promoter to drive expression of the fusion protein. Plasmids were transfected into 293T cells using Lipofectamin 2000 reagent according to the manufacturer's instructions (Invitrogen). Cells were cultured for 2 d in DMEM medium, and lysed in 0.1% NP-40 buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, and complete protease inhibitors [Amersham]). Lysates were centrifuged at 14,000g, before purification of protein complexes using glutathione Sepharose beads. Purified complexes and control lysate samples were run on Nu-PAGE acrylamide gels (Invitrogen), and Myc-and GST-tagged proteins were detected using standard immunoblotting techniques. Mouse monoclonal anti-Myc (clone 9E10) and rabbit polyclonal anti-GST were purchased from Sigma.
Immunofluorescence and imaging
Immunostaining was performed essentially as described (Page et al. 2001). Briefly, dissected gonads were fixed in 1% paraformaldehyde in PBS and permeabilized in TBS supplemented with 0.1% Triton X-100. The samples were subsequently incubated with the following antibodies: rabbit anti-GLA-3A 1301, 1:1000 or rabbit anti-GLA-3B 1297, 1:1000 in 5% BSA in TBS, at 4°C overnight. Anti-rabbit AlexaFluor 594 or anti-mouse Alexa-Fluor 594 IgG antibodies were used as secondary antibodies (Molecular Probes). DAPI was added to a final concentration of 200 ng/mL, and samples were mounted for microscopy. All images were analyzed by light microscopy with a Zeiss Axioskop equipped with epifluorescence and differential interference contrast (DIC) optics. Digital images were acquired and processed using a CCD camera and Openlab software. Histochemical staining of 
-galactosidase with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) was performed as described (Zdinak et al. 1997).
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6 Present address: Laboratoire génomique virale et vaccination, CNRS UMR1930, Institut Pasteur de Paris, 28 Rue du Docteur Roux, Paris, 75724 Cedex 15, France.
E-MAIL michael.hengartner{at}molbio.unizh.ch; FAX 41-44-635-6861.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.384506.
Supplemental material is available at http://www.genesdev.org.
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